Method for improving vegf-receptor blocking selectivity of an anti-vegf antibody

ABSTRACT

The present invention relates to methods for improving anti-VEGF antibodies in order to provide or improve antibodies that preferentially inhibit binding to VEGF to VEGF-R2 rather than VEGF binding to VEGF-R1; and antibodies provided by said methods.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/EP2018/086468, filed Dec. 21, 2018, the disclosure of which isincorporated herein by reference in its entirety, and which claimspriority to European Patent Application No. 17211032.2 filed Dec. 29,2017.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 12, 2020, isnamed P35212_US_SequenceListing.txt and is 42.7 kilobytes in size.

FIELD OF THE INVENTION

The present invention relates to methods for improving anti-VEGFantibodies in order to provide or improve antibodies that preferentiallyinhibit binding to VEGF to VEGF-R2 rather than VEGF binding to VEGF-R1;and antibodies provided by said methods.

BACKGROUND OF THE INVENTION

Anti-VEGF antibodies that are approved for clinical application, such asAvastin® and Lucentis®, inhibit VEGF-binding to both receptors, VEGF-R1(FLT-1, fms-like tyrosine kinase) and VEGF-R2 (KDR/FLK-1, fetal liverkinase). VEGF-R1 and VEGF-R2 are closely related receptor tyrosinekinases (RTK). While VEGF-R2 is hypothized to be primarily responsiblefor VEGF-mediated angiogenesis (Holash, J. et al., Proc Natl Acad SciUSA. 2002 Aug. 20; 99(17):11393-8), VEGF-R1 is known to have otherimportant biological roles unrelated to angiogenesis, e.g. in osteoclastdifferentiation (Aldridge, S. E. et al., Biochem Biophys Res Commun.2005 Sep. 30; 335(3):793-8).

A few anti-VEGF antibodies that preferentially inhibit VEGF binding toVEGF-R2 and but do not significantly inhibit VEGF-binding to VEGF-R1have been reported, but were not yet clinically successfull (WO200064946describing an antibody termed “2C3”, WO 2009060198 describing anantibody termed “r84”, and WO 2012089176 describing an antibody termed“L3H6”, EP3006465 describing antibodies termed “HF2-1, HF2-5, HF2-9, andHF2-11”). By blocking VEGF binding to VEGF-R2, but not VEGF-R1, theantibodies are described to have an improved safety profile and do notshow common toxicity-related side effects associated with anti-VEGFtherapy (Brekken, R. A., et al., Cancer Res. 2000 Sep. 15;60(18):5117-24; Sullivan, L. A., et al., PLoS One, 2010 Aug. 6;5(8):e12031).

There is a need for methods for improving anti-VEGF antibodies,particularly the VEGFR-blocking selectivity, to provide promisingclinical antibody candidates.

SUMMARY OF THE INVENTION

The present invention relates to a method of improving VEGFR-blockingselectivity of an antibody that binds to VEGF comprising an antigenbinding site formed by cognate pair of a VH and a VL domain, wherein theantibody binds to an epitope of VEGF that overlaps with theVEGF-R1-binding region and the VEGF-R2-binding region in the VEGFmolecule. The method of the invention comprises (a) providing ananalysis of the tertiary structure of a complex of a VEGF-dimer bound bya first and a second antigen binding site of said antibody that binds toVEGF (VEGF-dimer-antibody-complex), (b) identifying at least one aminoacid residue located in the VH domain or VL domain of said antibody,wherein said amino acid residue within the first antigen binding siteand said amino acid residue within the second antigen binding site arespatially arranged in close proximity in the VEGF-dimer-antigen-complex;and (c) substituting said at least one amino acid residue identified instep b) by an amino acid having a larger side chain volume.

With the method of the invention the VEGFR-blocking selectivity ofcertain anti-VEGF antibodies may be improved by a few modifications intheir amino acid sequence, particularly in regions that are not involvedin antigen binding. Such antibodies may exhibit an improved safetyprofile, e.g. by avoiding or reducing side effects caused by blockingVEGF-signalling through VEGF-R1.

In one embodiment the antibody binds to a conformational epitope on adimer of VEGF-A121, wherein VEGF-A121 comprises an amino acid sequenceof SEQ ID NO: 36, wherein the epitope comprises in one of the individualVEGF-A121 molecules within the VEGF dimer amino acids F17, M18, D19,Y21, Q22, R23, Y25, H27, P28, 129, E30, M55, N62, L66, N100, K101, C102,E103, C104, R105 and P106; and in the other one of the individualVEGF-A121 molecules within the VEGF dimer amino acids E30, K48, M81 andQ87. In one embodiment the epitope is measured by x-ray crystallography.

In one embodiment the amino acid with a larger side chain volume is anaromatic amino acid.

In one embodiment the at least one substituted amino acid residue islocated in the heavy chain variable domain of said antibody.

Another aspect of the invention is an antibody that binds to VEGFprovided by a method of one of the invention.

Another aspect of the invention is antibody that binds to VEGF, whereinbinding of the antibody to VEGF significantly inhibits VEGF-binding toVEGF receptor VEGF-R2 without significantly inhibiting VEGF-binding toVEGF receptor VEGF-R1, provided by a method of the invention.

DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1: Crystal structure of VEGF dimer (purple) in complex with VEGF-R1domain 2 (red) and with VEGF-R2 domain 2 and 3 (blue)

FIG. 2: Inhibition of VEGF-binding to VEGF-R1 and VEGF-R2 in presence ofantibody Fab fragments (VEGF:VEGF-R2/R1 inhibitition ELISA) as describedin Example 2.

FIG. 3: Crystal structure of VEGF dimer (purple) in complex withanti-VEGF antibody VEGF-0089 as determined by X ray crystallographyaccording to Example 3. Red circle highlights regions in the VH domainof VEGF-0089 that are in close proximity.

FIG. 4: Epitope amino acids bound by VEGF-0089 Fab fragment in a dimerof VEGF-A121 (SEQ ID NO: 36) as determined by X ray crystallographyaccording to Example 3. Amino acid positions comprised in each one ofthe VEGF-A121 molecules in contact with VEGF-0089 Fab fragment within adistance of 5 Å are highlighted in black.

FIG. 5: Overlay of the crystal structures of a human VEGF-A121-dimer incomplex with VEGF-R1 domain 2 and a human VEGF-A121-dimer in complexwith VEGF-0089 Fab as measured in Example 3.

FIG. 6: Overlay of the crystal structures of a human VEGF-A121-dimer incomplex with VEGF-R2 domains 2 and 3 and a human VEGF-A121-dimer incomplex with VEGF-0089 Fab as measured in Example 3.

FIG. 7: Inhibition of VEGF binding to VEGF-R1 in presence of anti-VEGFantibodies as described in Example 4 (0.34 nM VEGF).

FIG. 8: Inhibition of VEGF binding to VEGF-R1 in presence of anti-VEGFantibodies as described in Example 4 (0.7 nM VEGF).

FIG. 9: Inhibition of VEGF binding to VEGF-R2 in presence of anti-VEGFantibodies as described in Example 4 (0.34 nM VEGF).

FIG. 10: Inhibition of VEGF binding to VEGF-R2 in presence of anti-VEGFantibodies as described in Example 4 (0.7 nM VEGF).

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The methods andtechniques of the present disclosure are generally performed accordingto conventional methods well known in the art. Generally, nomenclaturesused in connection with, and techniques of biochemistry, enzymology,molecular, and cellular biology, microbiology, genetics and protein andnucleic acid chemistry and hybridization described herein are thosewell-known and commonly used in the art.

Unless otherwise defined herein the term “comprising of” shall includethe term “consisting of”.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments so long as they exhibit the desired antigen-bindingactivity.

Papain digestion of intact antibodies produces two identicalantigen-binding fragments, called “Fab” fragments containing each theheavy- and light-chain variable domains (VH and VL, respectively) andalso the constant domain of the light chain (CL) and the first constantdomain of the heavy chain (CH1). The term “Fab fragment” thus refers toan antibody fragment comprising a light chain comprising a VL domain anda CL domain, and a heavy chain fragment comprising a VH domain and a CH1domain.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC) methods. For a review of methods for assessment of antibodypurity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)2; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs), including thecomplementarity determining regions (CDRs) (see, e.g., Kindt et al. KubyImmunology, 6th ed., W.H. Freeman and Co., page 91 (2007)).

A “paratope” or an “antigen binding site”, as used interchangeablyherein, refers to a part of an antibody which recognizes and binds to anantigen. An antigen binding site is formed by several individual aminoacid residues from the antibody's heavy and light chain variable domainsarranged that are arranged in spatial proximity in the tertiarystructure of the Fv region. In one embodiment, the antigen binding siteis defined as a set of the six CDRs comprised in a cognate VH/VL pair.

The term “complementarity determining regions” or “CDRs” as used hereinrefers to each of the regions of an antibody variable domain which arehypervariable in sequence and contain antigen-contacting residues.Generally, antibodies comprise six CDRs: three in the VH domain (CDR-H1,CDR-H2, CDR-H3), and three in the VL domain (CDR-L1, CDR-L2, CDR-L3).Unless otherwise indicated, CDR residues and other residues in thevariable domain (e.g., FR residues) are numbered herein according to theKabat numbering system (Kabat et al., Sequences of Proteins ofImmunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md., 1991).

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below.

“Framework” or “FR” as used herein refers to variable domain amino acidresidues other than CDR residues. The framework of a variable domaingenerally consists of four framework domains: FR1, FR2, FR3, and FR4.Accordingly, the CDR and FR amino acid sequences generally appear in thefollowing sequence in the (a) VH domain:FR1-CDR-H1-FR2-CDR-H2-FR3-CDR-H3-FR4; and (b) in the VL domain:FR1-CDR-L1-FR2-CDR-L2-FR3-CDR-L3-FR4.

Vascular endothelial growth factor (VEGF) is a homodimeric member of thecystine knot family of growth factors. The term “VEGF”, as used herein,refers to any native VEGF from any vertebrate source, including mammalssuch as primates (e.g. humans) and rodents (e.g., mice and rats), unlessotherwise indicated. The term encompasses “full-length”, unprocessedVEGF as well as any form of VEGF that results from processing in thecell. The term also encompasses naturally occurring variants of VEGF,e.g., splice variants or allelic variants. The amino acid sequence of anexemplary human VEGF is shown in SEQ ID NO:33. The term “VEGF-dimer” asused to herein refers to a homodimer of two identical VEGF-molecules. Acomplex formed by two identical antibody molecules that are bound to aVEGF-dimer is herein referred to as “VEGF-dimer-antibody-complex”.

A “first and a second antigen binding site” comprised in aVEGF-dimer-antibody-complex refers to the antigen binding site that iscomprised in the VH/VL pair of each one of the two antibodies comprisedin the VEGF-dimer-antibody-complex. For example, while the antigenbinding site of one of the two anti-VEGF antibodies in theVEGF-dimer-antibody-complex is the “first antigen binding site”, theantigen binding site of other one of the two anti-VEGF antibodies isautomatically the “second antigen binding site”.

VEGF stimulates cellular responses by binding to tyrosine kinasereceptors (the VEGF-receptors, or “VEGFRs”) on the cell surface, causingthem to dimerize and become activated through transphosphorylation,although to different sites, times, and extents. VEGF-R1 and VEGF-R2 areclosely related receptor tyrosine kinases (RTK). VEGF-A binds to VEGFR-1(Flt-1), interacting with domain 2 of VEGF-R1, and VEGFR-2 (KDR/Flk-1),interacting with domains 2 and 3 of VEGF-R2 (see FIG. 1).

The “VEGF-R1-binding region” and “VEGF-R2-binding region” of a VEGFmolecule or a VEGF-dimer as used herein refers to those amino acids onthe VEGF that interact with domain 2 of VEGF-R1 or domains 2 or 3 ofVEGF-R2, respectively.

The terms “anti-VEGF antibody” and “an antibody that binds to VEGF”refer to an antibody that is capable of binding VEGF with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting VEGF. In one embodiment, the extent ofbinding of an anti-VEGF antibody to an unrelated, non-VEGF protein isless than about 10% of the binding of the antibody to VEGF as measured,e.g., by surface plasmon resonance (SPR). In certain embodiments, anantibody that binds to VEGF has a dissociation constant (K_(D)) of <1nM, or <0.15 nM. An antibody is said to “specifically bind” to VEGF whenthe antibody has a K_(D) of 1 μM or less.

By “VEGFR-blocking selectivity” is used herein as an abbreviative termwhen referred to the property of anti-VEGF antibodies thatpreferentially inhibit binding to VEGF to VEGF-R2 rather than VEGFbinding to VEGF-R1, when bound to a VEGF-dimer. Anti-VEGF antibodiesthat are capable of fully blocking VEGF-binding to VEGF-R2, but notfully block VEGF-binding to VEGF-R1, are condifered to selectively blockVEGF-signalling through VEGF-R2 but not through VEGF-R1, i.e. exhibit“VEGFR-blocking selectivity”.

The “tertiary structure” of a proteinis the three dimensional shape ofthe protein. The tertiary structure exhibits a single polypeptide chain“backbone” with one or more protein secondary structures, the proteindomains. Amino acid side chains may interact and bond in a number ofways. The interactions and bonds of side chains within a particularprotein determine its tertiary structure. The “tertiary structure of aVEGF-dimer-antibody-complex” as used herein means the threedimensionalshape of said complex.

Amino acid residues located “in close proximity” within a tertiarystructure of a VEGF-dimer-antibody-complexes are amino acid residuesderived from both anti-VEGF antibodies that are spatially arranged inthe threedimensional shape of said complex in a way that their distanceis up to 5 Å. This does not include amino acids adjacent to each otherin the amino acid sequence of the individual domain, i.e. VH or VL, ofthe respective anti-VEGF antibody.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (K_(D)). Affinity can be measured by common methods known inthe art, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are describedherein.

The term “epitope” denotes the site on an antigen, either proteinaceousor non-proteinaceous, to which an anti-VEGF antibody binds. Epitopes canbe formed both from contiguous amino acid stretches (linear epitope) orcomprise non-contiguous amino acids (conformational epitope), e.g.coming in spatial proximity due to the folding of the antigen, i.e. bythe tertiary folding of a proteinaceous antigen. Linear epitopes aretypically still bound by an anti-VEGF antibody after exposure of theproteinaceous antigen to denaturing agents, whereas conformationalepitopes are typically destroyed upon treatment with denaturing agents.An epitope comprises at least 3, at least 4, at least 5, at least 6, atleast 7, or 8-10 amino acids in a unique spatial conformation.

Screening for antibodies binding to a particular epitope (i.e., thosebinding to the same epitope) can be done using methods routine in theart such as, e.g., without limitation, alanine scanning, peptide blots(see Meth. Mol. Biol. 248 (2004) 443-463), peptide cleavage analysis,epitope excision, epitope extraction, chemical modification of antigens(see Prot. Sci. 9 (2000) 487-496), and cross-blocking (see “Antibodies”,Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harb., NY).

Antigen Structure-based Antibody Profiling (ASAP), also known asModification-Assisted Profiling (MAP), allows to bin a multitude ofmonoclonal antibodies specifically binding to VEGF based on the bindingprofile of each of the antibodies from the multitude to chemically orenzymatically modified antigen surfaces (see, e.g., US 2004/0101920).The antibodies in each bin bind to the same epitope which may be aunique epitope either distinctly different from or partially overlappingwith epitope represented by another bin.

Also competitive binding can be used to easily determine whether anantibody binds to the same epitope of VEGF as, or competes for bindingwith, a reference anti-VEGF antibody. For example, an “antibody thatbinds to the same epitope” as a reference anti-VEGF antibody refers toan antibody that blocks binding of the reference anti-VEGF antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. Also for example, to determine if anantibody binds to the same epitope as a reference anti-VEGF antibody,the reference antibody is allowed to bind to VEGF under saturatingconditions. After removal of the excess of the reference anti-VEGFantibody, the ability of an anti-VEGF antibody in question to bind toVEGF is assessed. If the anti-VEGF antibody is able to bind to VEGFafter saturation binding of the reference anti-VEGF antibody, it can beconcluded that the anti-VEGF antibody in question binds to a differentepitope than the reference anti-VEGF antibody. But, if the anti-VEGFantibody in question is not able to bind to VEGF after saturationbinding of the reference anti-VEGF antibody, then the anti-VEGF antibodyin question may bind to the same epitope as the epitope bound by thereference anti-VEGF antibody. To confirm whether the antibody inquestion binds to the same epitope or is just hampered from binding bysteric reasons routine experimentation can be used (e.g., peptidemutation and binding analyses using ELISA, RIA, surface plasmonresonance, flow cytometry or any other quantitative or qualitativeantibody-binding assay available in the art). This assay should becarried out in two set-ups, i.e. with both of the antibodies being thesaturating antibody. If, in both set-ups, only the first (saturating)antibody is capable of binding to VEGF, then it can be concluded thatthe anti-VEGF antibody in question and the reference anti-VEGF antibodycompete for binding to VEGF.

In some embodiments two antibodies are deemed to bind to the same or anoverlapping epitope if a 1-, 5-, 10-, 20- or 100-fold excess of oneantibody inhibits binding of the other by at least 50%, at least 75%, atleast 90% or even 99% or more as measured in a competitive binding assay(see, e.g., Junghans et al., Cancer Res. 50 (1990) 1495-1502).

In some embodiments two antibodies are deemed to bind to the sameepitope if essentially all amino acid mutations in the antigen thatreduce or eliminate binding of one antibody also reduce or eliminatebinding of the other. Two antibodies are deemed to have “overlappingepitopes” if only a subset of the amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding” an antibody refers to one or morenucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “vector”, as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors”.

The terms “host cell”, “host cell line”, and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells”, which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

Amino acids may be grouped according to common side-chain properties:(1) hydrophobic side chains: Norleucine, Met (M), Ala (A), Val (V), Leu(L), Ile (I); uncharged hydrophilic side chains (also referred to in theart as “neutral” hydrophilic side chains): Cys (C), Ser (S), Thr (T),Asn (N), Gln (Q); negatively charged side chains (also referred to inthe art as “acidic” side chains): Asp (D), Glu (E); positively chargedside chains (also referred to in the art as “basic” side chains): His(H), Lys (K), Arg (R); aromatic side chains: Trp (W), Tyr (Y), Phe (F);and side chains that comprise residues that influence chain orientation:Gly (G), Pro (P).

When referring to amino acid modifications, amino acids “having a largerside chain volume” refer to amino acids that have a larger side chainvolume than the original amino acid located at the position to bemodified. In certain embodiments, amino acids having a larger side chainvolume are aromatic amino acids, including tryptophane, tyrosine andphenylalanine.

2. Detailed Description of the Embodiments of the Invention

The present invention relates to a method of improving VEGFR-blockingselectivity of an antibody that binds to VEGF comprising an antigenbinding site formed by cognate pair of a VH and a VL domain, wherein theantibody binds to an epitope of VEGF that overlaps with theVEGF-R1-binding region and the VEGF-R2-binding region in the VEGFmolecule. The method of the invention comprises (a) providing ananalysis of the tertiary structure of a complex of a VEGF-dimer bound bya first and a second antigen binding site of said antibody that binds toVEGF (VEGF-dimer-antibody-complex), (b) identifying at least one aminoacid residue located in the VH domain or VL domain of said antibody,wherein said amino acid residue within the first antigen binding siteand said amino acid residue within the second antigen binding site arespatially arranged in close proximity in the VEGF-dimer-antigen-complex;and (c) substituting said at least one amino acid residue identified instep (b) by an amino acid having a larger side chain volume. With amethod of the invention an antibody is provided that preferentiallyinhibits binding to VEGF to VEGF-R2 rather than VEGF binding to VEGF-R1(VEGFR-blocking selectivity).

The method of the invention is for improving anti-VEGF antibodies thatbind to an epitope of VEGF that overlaps with the VEGF-R1-binding regionand the VEGF-R2-binding region in the VEGF molecule. In one embodimentsaid epitope comprises amino acids interacting with domain 2 of VEGF-R1,when VEGF is bound to VEGF-R1. In one embodiment said epitope comprisesamino acids interacting with domains 2 and 3 of VEGF-R2, when VEGF isbound to VEGF-R1. In one embodiment said anti-VEGF antibody binds to anepitope that overlaps with the epitope bound by an antibodycharacterized by a VH of SEQ ID NO:01 and a VL of SEQ ID NO:02 (antibodyVEGF-0089 as described herein). In one embodiment said anti-VEGFantibody binds to the same epitope than antibody VEGF-0089 as describedherein, as measured by x-ray crystallography. In one embodiment saidanti-VEGF antibody binds to the same epitope than antibody VEGF-0089 asdescribed herein, as measured by x-ray crystallography as described inExample 3. In one embodiment, said epitope is a conformational epitopewithin a dimer of VEGF-A121, wherein VEGF-A121 comprises an amino acidsequence of SEQ ID NO: 36, wherein the epitope comprises in one of theindividual VEGF-A121 molecules within the VEGF dimer amino acids F17,M18, D19, Y21, Q22, R23, Y25, H27, P28, 129, E30, M55, N62, L66, N100,K101, C102, E103, C104, R105 and P106; and in the other one of theindividual VEGF-A121 molecules within the VEGF dimer amino acids E30,K48, M81 and Q87. The numbering is according to the position of theamino acid in the amino acid sequence of VEGF-A121 indicated in SEQ IDNO: 36 (see also FIG. 4).

Within a method of the invention an analysis of the tertiary structureof a VEGF-dimer-antibody-complex is provided. The tertiary structure maybe provided by methods known in the art. In one embodiment the tertiarystructure is provided by x-ray crystallography, e.g. as described inExample 3 herein.

In a method of the invention amino acid residues in the VH domain and/orVL domain of said antibody are identified that are in close proximitywithin the VEGF-dimer-antibody-complex. In one embodiment “closeproximity” refers to a distance of 10 Å or less. In one embodiment“close proximity” refers to a distance of 5 Å or less. This at least oneamino acid residue is considered suitable for modification.

In one embodiment such at least one amino acid residue identified instep (b) is located in the heavy chain variable domain of said antibody.In one embodiment the at least one amino acid residue identified in step(b) is located within a heavy chain CDR of said antibody. In oneembodiment the at least one amino acid residue identified in step (b) islocated within H-CDR2 of said antibody. In one embodiment the at leastone amino acid residue identified in step (b) is located within a heavychain FR of said antibody. In one embodiment the at least one amino acidresidue identified in step (b) is located within H-FR3 of said antibody.

In the method of the invention such amino acid residue is substituted byan amino acid having a larger side chain volume than the amino acidresidue comprised in the anti-VEGF antibody to be improved. In oneembodiment said amino acid having a larger side chain volume is anaromatic amino acid. In one embodiment said amino acid having a largerside chain volume is Trp (W), Tyr (Y), or Phe (F).

In one embodiment said anti-VEGF antibody of step a) binds to humanVEGFA. In one embodiment said anti-VEGF antibody of step a) binds tohuman VEGF of SEQ ID NO:33.

In one aspect the anti-VEGF antibody improved by a method of theinvention comprises (a) CDR-H1 comprising the amino acid sequence of SEQID NO:03; (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO:04;(c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:05; (d)CDR-L1 comprising the amino acid sequence of SEQ ID NO:06; (e) CDR-L2comprising the amino acid sequence of SEQ ID NO:07; and (f) CDR-L3comprising the amino acid sequence of SEQ ID NO:08. One exemplaryantibody comprising this set of CDR amino acid sequences is the antibodyreferred to herein as “VEGF-0089”. In one embodiment said antibody ischaracterized by a VH comprising SEQ ID NO:01 and a VL comprising SEQ IDNO:02.

In one embodiment of this aspect, the at least one amino acid residueidentified in step (b) is located within H-CDR2 of said antibody. In oneembodiment said at least one amino acid residue is selected from Kabatposition 52a, 52c and 54. In one embodiment one, two or three aminoacids of Kabat position 52a, 52c and 54 are modified. In one embodimentat least one amino acid residue is selected from Kabat position 52a, 52cand 54 is modified by substitutions with Trp (W), Tyr (Y), or Phe (F).In one embodiment step c) includes one or more of the followingsubstitutions N52aS, G52cP and I54F.

In one embodiment of this aspect, the at least one amino acid residueidentified in step (b) is located within H-FR3 of said antibody. In oneembodiment said at least one amino acid residue is Kabat position 74. Inone embodiment at least the amino acid residue at Kabat position 74 ismodified by substitutions with Trp (W), Tyr (Y), or Phe (F). In oneembodiment step c) includes a substitution S74W.

The present invention also relates to an anti-VEGF antibody provided bya method of the invention. The present invention further relates to ananti-VEGF antibody, wherein binding of the antibody to VEGFsignificantly inhibits VEGF-binding to VEGF receptor VEGF-R2 withoutsignificantly inhibiting VEGF-binding to VEGF receptor VEGF-R1, providedby a method of the invention. In one embodiment the antibody provided bya method of the invention is an isolated antibody.

Such anti-VEGF antibodies may be produced using recombinant methodsknown in the art, e.g., as described in U.S. Pat. No. 4,816,567, e.g.recombinant expression in eukaryotic cell such as HEK293 cells asdescribed in Example 1. For these methods one or more isolated nucleicacid(s) encoding an antibody are provided.

In certain embodiments, an antibody provided herein is an antibodyfragment. In one embodiment, the antibody fragment is a Fab, Fab′,Fab′-SH, or F(ab′)2 fragment, in particular a Fab fragment.

In certain embodiments, an antibody provided herein is a full lengthantibody.

In one embodiment the antibody is an IgG1 antibody.

The present invention further relates to a nucleic acid encoding for ananti-VEGF antibody provided by a method of the invention. The presentinvention further relates to a host cell comprising the nucleic acid ofthe invention.

DESCRIPTION OF THE AMINO ACID SEQUENCES SEQ ID NO: 1VH domain of antibody VEGF-0089EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPGKGLEWVSSIGNGGGIYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSS SEQ ID NO: 2VL domain of antibodies VEGF-0089, VEGF-0113,VEGF-0114, VEGF-P1AD8675, VEGF-P1AE3520, VEGF-P1AE3521, VEGF-0112,VEGF-P1AE8674, VEGF-P1AE3519 DIQMTQSPASLSASVGDRVTITCRASQSIYSSLNWYQQKPGKAPKLLIYASTLQSGVPSRFSGSASGTDFTLTISSLQPEDVAT YYCQQYQNFPRTFGQGTKLEIKSEQ ID NO: 3 H-CDR1 of antibodies VEGF-0089, VEGF-0113,VEGF-0114, VEGF-P1AD8675,  VEGF-P1AE3520, VEGF-P1AE3521,VEGF-0112, VEGF-P1AE8674, VEGF-P1AE3519 NYAMT SEQ ID NO: 4H-CDR2 of antibodies VEGF-0089, VEGF-P1AD8675, VEGF-P1AD8674 SIGNG GGIYTYYADSVKG SEQ ID NO: 5 H-CDR3 of antibodies VEGF-0089, VEGF-0113,VEGF-0114, VEGF-P1AD8675, VEGF-P1AE3520, VEGF-P1AE3521,VEGF-0112, VEGF-P1AE8674, VEGF-P1AE3519 GDNLFDS SEQ ID NO: 6L-CDR1 of antibodies VEGF-0089, VEGF-0113, VEGF-0114, VEGF-P1AD8675, VEGF-P1AE3520, VEGF-P1AE3521,VEGF-0112, VEGF-P1AE8674, VEGF-P1AE3519RASQSIYSSLN SEQ ID NO: 7 L-CDR2 of antibodies VEGF-0089, VEGF-0113,VEGF-0114, VEGF-P1AD8675, VEGF-P1AE3520, VEGF-P1AE3521,VEGF-0112, VEGF-P1AE8674, VEGF-P1AE3519 ASTLQSGVPSR SEQ ID NO: 8L-CDR3 of antibodies VEGF-0089, VEGF-0113, VEGF-0114, VEGF-P1AD8675,VEGF-P1AE3520, VEGF-P1AE3521, VEGF-0112, VEGF-P1AE8674, VEGF-P1AE3519FPRT SEQ ID NO: 9 H-FR3 of VEGF-0089, VEGF-0113,VEGF-0114, VEGF-0112FTISRDN S KNTLYLQMNSLRAEDTAVYYCAK SEQ ID NO: 10heavy chain of VEGF-0089 Fab fragmentEVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPGKGLEWVSSIGNGGGIYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDK TVAPSTCSEQKLISEEDLGAAEPEASEQ ID NO: 11 light chain of VEGF-0089, VEGF-0113,VEGF-0114, VEGF-P1AD8675 Fab fragmentDIQMTQSPASLSASVGDRVTITCRASQSIYSSLNWYQQKPGKAPKLLIYASTLQSGVPSRFSGSASGTDFTLTISSLQPEDVATYYCQQYQNFPRTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC SEQ ID NO: 12VH domain of antibody VEGF-0113EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPGKGLEWVSSIGNGPGIYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSS SEQ ID NO: 13H-CDR2 of antibody VEGF-0113, P1AE3520 SIGNGPGIYTYYADSVKG SEQ ID NO: 14heavy chain of VEGF-0113 Fab fragmentEVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPGKGLEWVSSIGNGPGIYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDK TVAPSTCSEQKLISEEDLGAAEPEASEQ ID NO: 15 VH domain of antibody VEGF-0114EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPGKGLEWVSSIGSGGFYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSS SEQ ID NO: 16H-CDR2 of antibody VEGF-0114, VEGF-P1AE3521 SIG S GG -F YTYYADSVKGSEQ ID NO: 17 heavy chain of VEGF-0114 Fab fragmentEVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPGKGLEWVSSIGSG_GFYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDK TVAPSTCSEQKLISEEDLGAAEPEASEQ ID NO: 18 VH domain of antibody VEGF-P1AD8675EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPGKGLEWVSSIGNGGGIYTYYADSVKGRFTISRDNWKNTLYLQMNSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSS SEQ ID NO: 19H-FR3 of antibody VEGF-P1AD8675, P1AE3520, VEGF-P1AE3521 FTISRDN WKNTLYLQMNSLRAEDTAVYYCAK SEQ ID NO: 20heavy chain of VEGF-P1AD8675 Fab fragmentEVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPGKGLEWVSSIGNGGGIYTYYADSVKGRFTISRDNWKNTLYLQMNSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSSASTKGPSVFPLAPSSKSISGGTAALGCLVKDYFPEPVTVSWNSGALISGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSC SEQ ID NO: 21VH domain of antibody VEGF-P1AE3520EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPGKGLEWVSSIGNGPGIYTYYADSVKGRFTISRDNWKNTLYLQMNSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSS SEQ ID NO: 22heavy chain of VEGF-P1AE3520 Fab fragmentEVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPGKGLEWVSSIGNGPGIYTYYADSVKGRFTISRDNWKNTLYLQMNSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDK TVAPSTCSEQKLISEEDLGAAEPEASEQ ID NO: 23 VH domain of antibody VEGF-P1AE3521EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPGKGLEWVSSIGSGGFYTYYADSVKGRFTISRDNWKNTLYLQMNSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSS SEQ ID NO: 24heavy chain of VEGF-P1AE3521 Fab fragmentEVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPGKGLEWVSSIGSGGFYTYYADSVKGRFTISRDNWKNTLYLQMNSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKT VAPSTCSEQKLISEEDLGAAEPEASEQ ID NO: 25 VH domain of antibody VEGF-0112EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPGKGLEWVSSIGSGGGIYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSS SEQ ID NO: 26H-CDR2 of antibody VEGF-0112 SIGSGGGIYTYYADSVKG SEQ ID NO: 27heavy chain of VEGF-0112 Fab fragmentEVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPGKGLEWVSSIGSGGGIYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDK TVAPSTCSEQKLISEEDLGAAEPEASEQ ID NO: 28 VH domain of antibody VEGF-P1AD8674EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPGKGLEWVSSIGNGGGIYTYYADSVKGRFTISRNNAENTLYLQMNSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSS SEQ ID NO: 29H-FR3 of antibody VEGF-P1AD8674 FTISRNNAENTLYLQMNSLRAEDTAVYYCAKSEQ ID NO: 30 heavy chain of VEGF-P1AD8674 Fab fragmentEVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPGKGLEWVSSIGNGGGIYTYYADSVKGRFTISRNNAENTLYLQMNSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSSASTKGPSVFPLAPSSKSISGGTAALGCLVKDYFPEPVTVSWNSGALISGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTK VDKKVEPKSC SEQ ID NO: 31VH domain of antibody VEGF-P1AE3519EVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPGKGLEWVSSIGSGGGIYTYYADSVKGRFTISRDNWKNTLYLQMNSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSS SEQ ID NO: 32heavy chain of VEGF-P1AE3519 Fab fragmentEVQLVESGGGLVQPGGSLRLSCAASGFTFTNYAMTWVRQAPGKGLEWVSSIGSGGGIYTYYADSVKGRFTISRDNWKNTLYLQMNSLRAEDTAVYYCAKGDNLFDSWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDK TVAPSTCSEQKLISEEDLGAAEPEASEQ ID NO: 33 human VEGF MNFLLSWVHWSLALLLYLHHAKWSQAAPMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKKSVRGKGKGQKRKRKKSRYKSWSVYVGARCCLMPWSLPGPHPCGPCSERRKHLFVQDPQTCKCSCKNTD SRCKARQLELNERTCRCDKPRRSEQ ID NO: 34 heavy chain of Lucentis (ranibizumab)DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC SEQ ID NO: 35light chain of Lucentis (ranibizumab)EVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCSEQ ID NO: 36 Human VEGF-A121 APMAEGGGQNHHEVVKFMDVYQRSYCHPIETLVDIFQEYPDEIEYIFKPSCVPLMRCGGCCNDEGLECVPTEESNITMQIMRIKPHQGQHIGEMSFLQHNKCECRPKKDRARQEKCDKPRR

EXAMPLES

The following examples are provided to aid the understanding of thepresent invention, the true scope of which is set forth in the appendedclaims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

Example 1 Generation of Human Anti-VEGF Antibody (Antibody VEGF-0089)

An overlay of the crystal structures of a human VEGF-dimer in complexwith VEGF-R1 domain 2 and VEGF-R3 domains 2 and 3 is depicted in FIG. 1.This superimposition illustrates that both VEGF receptors bind to ahighly similar region on the VEGF dimer and that it therefore appearshighly challenging to generate antibodies that bind to VEGF that do notinhibit VEGF-binding to both receptors, VEGF-R1 and VEGF-R2, in the samefashion. In line with this, among the plurality of anti-VEGF antibodiesknown in the art, only few antibodies were reported to selectively blockVEGF-binding to VEGF-R2 rather than VEGF-binding to VEGF-R1.

Antibody VEGF-0089 as described herein was derived from Rocheproprietary transgenic rabbits, expressing a humanized antibodyrepertoire, upon immunization with a VEGF-derived antigen. Transgenicrabbits comprising a human immunoglobulin locus are reported in WO2000/46251, WO 2002/12437, WO 2005/007696, WO 2006/047367, US2007/0033661, and WO 2008/027986. The animals were housed according tothe Appendix A “Guidelines for accommodation and care of animals” in anAAALAC-accredited animal facility. All animal immunization protocols andexperiments were approved by the Government of Upper Bavaria (permitnumber 55.2-1-54-2532-90-14) and performed according to the GermanAnimal Welfare Act and the Directive 2010/63 of the European Parliamentand Council.

Immunization of Transgenic Rabbits

Briefly, rabbits (n=3), 12-16 week old, were immunized with recombinanthuman VEGF-121 protein coupled to keyhole limpet hemocyanin (KLH)(produced in house). All animals were immunized with 400 μg protein,emulsified with complete Freund's adjuvant (CFA), at day 0 byintradermal application, followed by 200 ug protein emulsion at weeks 1,2, 6, 11 and 14, by alternating intramuscular and subcutaneousinjections. Blood was taken at days 4, 5 and 6 post immunizations,starting from the 4th immunization onwards. Serum was prepared forimmunogen-specific rabbit-, and human-specific immunoglobulin titerdetermination by ELISA, and peripheral mononuclear cells were isolated,which were used as a source of antigen-specific B cells in the B cellcloning process.

B Cell Cloning from Transgenic Rabbits

Isolation of Rabbit Peripheral Blood Mononuclear Cells (PBMC):

Blood samples were taken of immunized rabbits. EDTA containing wholeblood was diluted twofold with 1×PBS (PAA) before density centrifugationusing lympholyte mammal (Cedarlane Laboratories) according to thespecifications of the manufacturer. The PBMCs were washed twice with1×PBS.

EL-4 B5 Medium:

RPMI 1640 (Pan Biotech) supplemented with 10% FCS (Pan Biotech), 2 mMGlutamin, 1% penicillin/streptomycin solution (Gibco), 2 mM sodiumpyruvate, 10 mM HEPES (PAN Biotech) and 0.05 mM b-mercaptoethanole(Invitrogen) was used.

Coating of Plates:

Sterile cell culture 6-well plates were coated with 2 μg/ml KLH incarbonate buffer (0.1 M sodium bicarbonate, 34 mMDisodiumhydrogencarbonate, pH 9.55) over night at 4° C. Plates werewashed in sterile PBS three times before use.

Depletion of Macrophages/Monocytes or of Human Fc Binders:

The PBMCs were seeded on sterile 6-well plates (cell culture grade) todeplete macrophages and monocytes through unspecific adhesion. Each wellwas filled at maximum with 4 ml medium and up to 6×10e6 PBMCs from theimmunized rabbit and were allowed to bind for 1 h at 37° C. in theincubator. The cells in the supernatant (peripheral blood lymphocytes(PBLs)) were used for the antigen panning step.

Immune Fluorescence Staining and Flow Cytometry:

The anti-IgG FITC (AbD Serotec) and the anti-huCk PE (Dianova) antibodywas used for single cell sorting. For surface staining, cells from thedepletion and enrichment step were incubated with the anti-IgG FITC andthe anti-huCk PE antibody in PBS and incubated for 45 min in the dark at4° C. After staining the PBMCs were washed two fold with ice cold PBS.Finally the PBMCs were resuspended in ice cold PBS and immediatelysubjected to the FACS analyses. Propidium iodide in a concentration of 5μg/ml (BD Pharmingen) was added prior to the FACS analyses todiscriminate between dead and live cells. A Becton Dickinson FACSAriaequipped with a computer and the FACSDiva software (BD Biosciences) wereused for single cell sort.

B-Cell Cultivation:

The cultivation of the rabbit B cells was performed by a methoddescribed by Seeber et al. (S Seeber et al. PLoS One 9 (2), e86184. 2014Feb. 4). Briefly, single sorted rabbit B cells were incubated in 96-wellplates with 200 μl/well EL-4 B5 medium containing Pansorbin Cells(1:100000) (Calbiochem), 5% rabbit thymocyte supernatant (MicroCoat) andgamma-irradiated murine EL-4 B5 thymoma cells (5×10e5 cells/well) for 7days at 37° C. in the incubator. The supernatants of the B-cellcultivation were removed for screening and the remaining cells wereharvested immediately and were frozen at −80° C. in 100 μl RLT buffer(Qiagen).

RNA encoding the V domains of the antibodies was isolated. Forrecombinant expression of the antibody PCR-products coding for VH or VLwere cloned as cDNA into expression vectors and transiently transformedinto HEK-293 cells.

From the screening an antibody comprising a VH domain of SEQ ID NO:01and a VL domain of SEQ ID NO:02 was selected. This antibody is hereinalso referred to as antibody “VEGF-0089”. For the subsequent analysesthe antibody VEGF-0089 was generated as a Fab fragment (herein referredto as “VEGF-0089 Fab fragment” or simply “VEGF-0089 Fab”) having thehuman VH and VL domains and rabbit derived constant domains of the lightchain (CLkappa) and heavy chain (CH1). The amino acid sequence of theheavy chain of VEGF-0089 Fab fragment is SEQ ID NO:10. The amino acidsequence of the light chain of VEGF-0089 Fab fragment is SEQ ID NO:11.

Example 2 Characterisation of Generated Human Anti-VEGF Antibody(Antibody VEGF-0089)

VEGF-binding of antibody VEGF-0089 Fab fragment was assessed by surfaceplasmon resonance (SPR) as described below.

Determination of Antibody Binding Affinity by Surface Plasmon Resonance(SPR)

An anti-His capturing antibody (GE Healthcare 28995056) was immobilizedto a Series S Sensor Chip C1 (GE Healthcare 29104990) using standardamine coupling chemistry resulting in a surface densitiy of 500-1000resonance units (RU). As running and dilution buffer, HBS-P+(10 mMHEPES, 150 mM NaCl pH 7.4, 0.05% Surfactant P20) was used, themeasurement temperature was set to 25° C. and 37° C., respectively.hVEGF-A121 was captured to the surface with resulting capture levelsranging from 5 to 35 RU. Dilution series of anti-VEGF antibodies(0.37-30 nM) were injected for 120 s, dissociation was monitored for atleast 600 s at a flow rate of 30 μl/min. The surface was regenerated byinjecting 10 mM Glycine pH 1.5 for 60 s. Bulk refractive indexdifferences were corrected by subtracting blank injections and bysubtracting the response obtained from the control flow cell withoutcaptured hVEGF-A121. Rate constants were calculated using the Langmuir1:1 binding model within the Biacore Evaluation software.

As a result, the KD of the VEGF-0089 Fab fragment was determined to be134 pM (at a temperature of 25° C.).

For further characterization of the antibody, inhibition of VEGF-bindingto its receptors VEGF-R1 and VEGF-R2 in presence of VEGF-0089 Fabfragment was assessed as described below:

Inhibition of VEGF-Binding to VEGF-R1 and VEGF-R2 in Presence ofAntibody Fab Fragments (VEGF:VEGF-R2/R1 Inhibition ELISA)

384 well streptavidin plates (Nunc/Microcoat #11974998001) were coatedwith 0.25 μg/mlbiotinylated VEGF-R1 or 0.5 μg/ml biotinylated VEGF-R2(inhouse production, each 25 μl/well in DPBS (1×) (PAN, #P04-36500)).Plates were incubated for 1 h at room temperature. In parallel,VEGF-121-His (inhouse production) at a concentration of 0.7 nM wasincubated with antibodies in different dilutions (12×1:2 dilution steps,starting with a concentration of 500 nM). This pre-incubation step wascarried out in 384 well PP plates (Weidmann medical technology,#23490-101) in 1×OSEP buffer (bidest water, 10×, Roche, #11 666 789001+0.5% Bovine Serum Albumin Fraction V, fatty acid free, Roche, #10735 086 001+0.05% Tween 20). Plates were incubated for 1 h at roomtemperature. After washing VEGF-R1/VEGF-R2 coated streptavidin plates 3times with 90 μl/well PBST-buffer (bidest water, 10×PBS Roche#11666789001+0.1% Tween 20), 25 μl of samples from the VEGF-antibodypre-incubation plate were transferred to coated strepavidin plates whichwere subsequenty incubated for 1 h at room temperature. After washing 3times with 90 μl/well PBST-buffer, 25 μl/well detection antibody (antiHis POD, Bethyl, #A190-114P, 1:12000) in 1×OSEP was added. Afterincubation for 1 h at room temperature plates were washed 3 times with90 μl PBST-buffer. 25 μl TMB (Roche, #11 835 033 001) was added to allwells simultaneously. After 10 min incubation at room temperature,signals were detected at 370 nm/492 nm on a Tecan Safire 2 Reader.

As a control and representative for a prior art anti-VEGF antibody thatis used in the clinic, Lucentis® (ranibizumab, heavy chain amino acidsequence of SEQ ID NO:34, light chain amino acid sequence of SEQ IDNO:35) was assessed under the same conditions. The results are shown inFIG. 2.

The results indicate that VEGF-0089 Fab fragment is capable of fullyblocking VEGF-binding to VEGF-R2. VEGF-0089 Fab fragment did not fullyblock VEGF-binding to VEGF-R1. Consequently, VEGF-0089 Fab fragment isconsidered to selectively block VEGF-signalling through VEGF-R2 but notthrough VEGF-R1. As illustrated in FIG. 2, prior art antibody Lucentis®is capable of fully blocking VEGF-binding to both receptors, VEGF-R2 andVEGF-R1.

Example 3

X-Ray Crystallography of Antibody VEGF-0089 in Complex with VEGF-Dimerand Epitope Determination

The crystal structure of VEGF-0089 Fab fragment as described above wasanalyzed according to standard methods known in the art.

X-ray crystallography of VEGF-0089 Fab fragment in complex withVEGF-A121 was performed as follows:

Complex Formation and Purification of the Dimeric ComplexVEGF-A121-VEGF-0089 Fab.

For complex formation the VEGF-0089 Fab fragment and human VEGF-A121(Peprotech) were mixed in a 1.1:1 molar ratio. After incubation for 16hours overnight at 4° C. the complex was purified via gelfiltrationchromatography on a Superdex200 (16/600) column in 20 mM IViES, 150 mMNaCl, pH6.5. Fractions containing the dimeric complex were pooled andconcentrated to 1.44 mg/ml.

Crystallization of Dimeric VEGF-A121-VEGF-0089 Fab Complex. Initialcrystallization trials were performed in sitting drop vapor diffusionsetups at 21° C. at a protein concentration of 11.5 mg/ml. Crystalsappeared within 1 day out of 0.1 M Tris pH 8.5, 0.2 M LiSO₄, 1.26 M(NH₄)₂SO₄. Plate shaped crystals grew in a week to a final size of150×100×30 μm. The crystals were directly harvested from the screeningplate without any further optimization steps.

Data Collection and Structure Determination.

For data collection crystals were flash cooled at 100K in precipitantsolution with addition of 15% ethylene glycol as cryoprotectant.Diffraction data were collected at a wavelength of 1.0000 Å using aPILATUS 6M detector at the beamline X10SA of the Swiss Light Source(Villigen, Switzerland). Data have been processed with XDS (Kabsch, W.Acta Cryst. D66, 133-144 (2010)) and scaled with SADABS (BRUKER). Thecrystals belong to the space group C2 with cell axes of a=227.61 Å,b=66.97 Å, c=218.31 Å, β=104.54° and diffract to a resolution of 2.17 Å.The structure was determined by molecular replacement with PHASER(McCoy, A. J, Grosse-Kunstleve, R. W., Adams, P. D., Storoni, L. C., andRead, R. J. J. Appl. Cryst. 40, 658-674 (2007)) using the coordinates ofa related in house structure of a Fab fragment and VEGF as searchmodels. Programs from the CCP4 suite (Collaborative ComputationalProject, Number 4 Acta Cryst. D50, 760-763 (1994)) and Buster (Bricogne,G., Blanc, E., Brandl, M., Flensburg, C., Keller, P., Paciorek, W.,Roversi, P., Sharff, A., Smart, O. S., Vonrhein, C., Womack, T. O.(2011). Buster version 2.9.5 Cambridge, United Kingdom: Global PhasingLtd) have been used to subsequently refine the data. Manual rebuildingof protein using difference electron density was done with COOT (Emsley,P., Lohkamp, B., Scott, W. G. and Cowtan, K. Acta Cryst D66, 486-501(2010)). Data collection and refinement statistics for both structuresare summarized in the following Table. All graphical presentations wereprepared with PYMOL (DeLano Scientific, Palo Alto, Calif., 2002).

TABLE Data collection and structure refinement statistics DataCollection Wavelength (Å)    1.0 Resolution¹ (Å)   49.49-2.17(2.27-2.17) Space group C2 Unit cell (Å, °)   227.61 66.97 218.31, 90.00  104.54 90.00 Unique reflections 168745 (21164) Multiplicity    3.45(3.43) Completeness (%)   99.8 (99.6) Mean I/σ(I)    8.36 (0.71) R-meas   0.073 (0.86) CC1/2    0.999 (0.364) Refinement Resolution¹ (Å)  49.49-2.17 (2.23-2.17) Reflections used in refinement 168674 (12361)Reflections used for R-free  8487 (617) R-work³    0.185 (0.262) R-free⁴   0.227 (0.287) Number of atoms  16966 Protein residues  1466 RMS bonds(Å)    0.010 RMS angles (°)    1.20 Ramachandran favored (%)   97.85Ramachandran outliers (%)    0.15 Rotamer outliers (%)    3.47Clashscore    2.39 Average B-factor (Å²)   65.89 protein   66.85 solvent  64.05 ¹Values in parentheses refer to the highest resolution bins.²R_(merge) = Σ|I−<I>|/ΣI where I is intensity. ³R_(work) =Σ|F_(o)−<F_(c)>|/ΣF_(o) where F_(o) is the observed and F_(c) is thecalculated structure factor amplitude. ⁴R_(free) was calculated based on5% of the total data omitted during refinement.

A schematic illustration of the crystal structure of two VEGF-0089 Fabfragments in complex with a human VEGF-A121 dimer is shown in FIG. 3.Amino acid residues in the VEGF-A121 dimer in contact within a distanceof 5 Å with antibody VEGF-0089 Fab fragment form the conformationalepitope bound by VEGF-0089 Fab on the VEGF-A121 dimer. The amino acidsequence of VEGF-A121 is SEQ ID NO: 36. An illustration of the aminoacids comprised in the epitope on both VEGF-A121 molecules in the VEGFdimer is highlighted in FIG. 4.

Antibody VEGF-0089 Fab binds to the following epitope on the VEGF-A121dimer:

-   -   in one of the individual VEGF-A121 molecules within the VEGF        dimer amino acids F17, M18, D19, Y21, Q22, R23, Y25, H27, P28,        129, E30, M55, N62, L66, N100, K101, C102, E103, C104, R105 and        P106; and    -   in the other one of the individual VEGF-A121 molecules within        the VEGF dimer amino acids E30, K48, M81 and Q87.

An overlay of the crystal structures of a human VEGF-dimer in complexwith VEGF-R1 domain 2 and VEGF-R3 domains 2 and 3 is depicted in FIG. 1.This superimposition illustrates that both VEGF receptors bind to ahighly similar region on the VEGF dimer.

An overlay of the crystal structures of a human VEGF-dimer in complexwith VEGF-R1 domain 2 and in complex with VEGF-0089 Fab fragment isdepicted in FIG. 5. This superimposition illustrates that the lightchain of the VEGF-0089 antibody of the invention superimposes withdomain 2 of VEGF-R1. An overlay of the crystal structures of a humanVEGF-dimer in complex with VEGF-R2 domains 2 and 3, and in complex withVEGF-0089 Fab fragment is depicted in FIG. 6. This superimpositionillustrates that the light chain of the VEGF-0089 antibody of theinvention superimposes with domain 2 of VEGF-R2.

Consequently, the x-ray data show that the epitope bound by theVEGF-0089 antibody overlaps with the region of VEGF that binds toVEGF-R1 and VEGF-R2. From the structural information provided theexpectation would be that binding of the VEGF-0089 antibody to VEGFwould result in a similar inhibition of both VEGF-R1 and VEGF-R2 bindingto VEGF. This is, surprisingly, not the case as demonstrated above inExample 2 and FIG. 2. Rather, antibody VEGF-0089 preferentially inhibitsbinding of VEGF to VEGF-R2 rather than the binding of VEGF to VEGF-R1.

Without being bound to this theory, this observation may be resultingfrom different affinities of VEGF towards its two receptors, VEGF-R1 andVEGF-R2. While VEGF-R1 is known to be bound by VEGF with strongaffinity, it is known that VEGF-R2 is known to be bound by VEGF withweak affinity. As VEGF exists in dimeric form, it is believed that thenumber of antibody molecules bound to the VEGF dimer influence bindingto VEGF-R1. It is believed that in case only one antibody molecule isbound to the VEGF dimer, the VEGF dimer is still capable of binding tothe strong affinity receptor VEGF-R1 while binding to the low affinityreceptor VEGF-R2 is inhibited.

Example 4 Provision of Improved Variants of VEGF-0089

Based on the theory described in Example 3, the VEGF-0089 antibody wasmodified in order to facilitate that preferentially only one antibodymolecule rather than two antibody molecules are capable ofsimultaneously binding to the VEGF dimer. From the tertiary structure ofthe VEGF-dimer-antibody-complex shown in FIG. 3 it can be seen that tworegions within the antibody's VH domain, i.e. H-CDR2 and H-FR3, are inclose spatial proximity to each other. To avoid simultaneous binding oftwo antibody molecules to the VEGF-dimer those regions were modified byamino acid substitutions using large amino acids to replace amino acidswith smaller side chain volume in this regions. In the examples aromaticamino acids were used to replace the amino acid comprised in theoriginal VEGF-0089 amino acid sequence. The theory was, that in thiscase binding of two antibody molecules to a VEGF dimer would besterically hindered and the observed preferential inhibition ofVEGF-binding to VEGF-R2 rather than the VEGF-binding to VEGF-R1 would beeven more prominent.

Based on this theory, the following antibody variants of antibodyVEGF-0089 were generated. Candidate antibodies VEGF-0013, VEGF-0014,VEGF-P1AD8675, VEGF-P1AE3520 and VEGF-P1AE3521 comprise amino acidsubstitutions with aromatic amino acid residues within H-CDR2, H-FR3 orboth. The listed control antibodies comprise amino acid substitutionsthat were expected to have no effect or to even have a detrimentaleffect on VEGFR-blocking selectivity.

TABLE 1 Amino acid sequences and amino acid modifications in generatedcandidate and control antibodies SEQ ID NO: H-CDR2 H-FR3 VII VLVEGF-0089 wt wt  1 2 Candidate antibodies VEGF-0113 G52cP wt 12 2VEGF-0114 N52aS, G52c(−), wt 15 2 I54F VEGF-P1AD8675 wt S74W 18 2VEGF-P1AE3520 G52cP S74W 21 2 VEGF-P1AE3521 N52aS, G52c(−), S74W 23 2I54F Control antibodies VEGF-0112 N52aS wt 25 2 VEGF-P1AD8674 wt D72N,S74A, 28 2 K75E VEGF-P1AE3519 N52aS S74W 31 2Fab fragments of the antibodies were cloned and expressed as describedin Example 1. Amino acid sequences of heavy chains and light chains areshown in Table 2.

TABLE 2 Amino acid sequences of anti-VEGF antibodies heavy chain lightchain VEGF-0089 Fab SEQ ID NO: 10 SEQ ID NO: 11 VEGF-0113 Fab SEQ ID NO:14 SEQ ID NO: 11 VEGF-0114 Fab SEQ ID NO: 17 SEQ ID NO: 11 VEGF-P1AD8675Fab SEQ ID NO: 20 SEQ ID NO: 11 VEGF-P1AE3520 Fab SEQ ID NO: 22 SEQ IDNO: 11 VEGF-P1AE3521 Fab SEQ ID NO: 24 SEQ ID NO: 11 VEGF-0112 Fab SEQID NO: 27 SEQ ID NO: 11 VEGF-P1AD8674 Fab SEQ ID NO: 30 SEQ ID NO: 11VEGF-P1AE3519 Fab SEQ ID NO: 32 SEQ ID NO: 11

Example 4 VEGFR-Blocking Selectivity of Improved Variants of VEGF-0089

Binding of VEGF to VEGF-R1 as well as VEGF-R2 in presence of improvedantibody Fab fragments was tested as described in Example 2. Allantibody Fab fragments listed in Table 2 were tested. Results are shownin FIGS. 7 to 10.

Example 5 VEGF-Binding Affinity of Improved Variants of VEGF-0089

The affinity of the antibodies was determined by SPR using the samemethods as described in Example 2. All antibody Fab fragments listed inTable 2 were tested. Results are shown in Table 3.

TABLE 3 Affinities of anti-VEGF antibodies KD [pM] at 25° C. KD [pM] at37° C. VEGF-0089 Fab 143 110 VEGF-0113 Fab  22  29 VEGF-0114 Fab  50  54VEGF-P1AD8675 Fab  88  65 VEGF-P1AE3520 Fab  31  39 VEGF-P1AE3521 Fab 32  61 VEGF-P1AE3519 Fab 119  94

Example 6 Chemical Stability of Improved Variants of VEGF-0089

Chemical stability of improved antibody Fab fragments was tested asfollows:

Chemical Degradation Test:

Antibody samples were formulated in 20 mM His/HisCl, 140 mM NaCl, pH6.0, and were split into three aliquots: one aliquot was re-bufferedinto PBS, respectively, and two aliquots were kept in the originalformulation. The PBS aliquot and one His/HisC1 aliquot were incubatedfor 2 weeks (2 w) at 40° C. (His/NaCl) or 37° C. (PBS) in 1 mg/ml, thePBS sample was incubated further for total 4 weeks (4 w). The thirdcontrol aliquot sample was stored at −80° C. After incubation ended,samples were analyzed for relative active concentration (Biacore; activeconcentration of both stressed aliquots of each binder is normalized tounstressed 4° C. aliquot), aggregation (SEC) and fragmentation(capillary electrophoresis or SDS-PAGE) and compared with the untreatedcontrol.

For Size Exclusion UHPLC (=SEC), proteins were separated depending ontheir molecular size in solution using a chromatographic gel like TSKgelUP-SW3000. With this method, protein solution was analyzed regardingtheir relative content of monomer, high molecular species (e.g.aggregates, dimers, impurities) and low molecular species (e.g.degradation products, impurities). 0.2 M Potassium phosphate, 0.25 MKCl, pH 6.2 was used as mobile phase. Protein solutions were dilutedsuch as ˜50μ of protein was injected in a volume of 5 and analyzed witha flow rate of 0.3 ml/min at 25° C. Protein detection was done at 280nm. Peak definition and peak integration were performed as demonstratedin the typical chromatograms in the product specific informationdocument.

All antibody Fab fragments listed in Table 2 were tested. Results areshown in Tables 4 and 5.

TABLE 4 VEGF-binding activity after stress of improved antibody Fabfragments 2 w/40° C./pH 6.0 2 w/37° C./pH 7.4 4 w/37° C./pH 7.4 VEGF-101 102 101 0089 Fab VEGF- 103 103 101 0113 Fab VEGF-  99 102  99 0114Fab VEGF- 101 102 109 P1AD8675 Fab VEGF- 100 102 101 P1AE3520 Fab VEGF-101 103  99 P1AE3521 Fab VEGF- 102 102 102 P1AE3519 Fab

TABLE 5 Molecular integrity after stress (4 weeks, pH 7.4, 37° C.) ofimproved antibody Fab fragments Aggregation Main fraction [% aggregates][%] VEGF-0089 Fab  0.6 99.4 VEGF-0113 Fab  0.6 99.4 VEGF-0114 Fab  297.1 VEGF-P1AD8675 Fab 65.3 34.7 VEGF-P1AE3520 Fab 11.9 87.2VEGF-P1AE3521 Fab  4.8 94.2 VEGF-P1AE3519 Fab  3 96.4

1. A method of improving VEGFR-blocking selectivity of an antibody thatbinds to VEGF comprising an antigen binding site formed by cognate pairof a VH and a VL domain, wherein the antibody binds to an epitope ofVEGF that overlaps with the VEGF-R1-binding region and theVEGF-R2-binding region in the VEGF molecule, the method comprising thesteps of: a) providing an analysis of the tertiary structure of acomplex of a VEGF-dimer bound by a first and a second antigen bindingsite of said antibody that binds to VEGF (VEGF-dimer-antibody-complex);b) identifying at least one amino acid residue located in the VH domainor VL domain of said antibody, wherein said amino acid residue withinthe first antigen binding site and said amino acid residue within thesecond antigen binding site are spatially arranged in close proximity inthe VEGF-dimer-antigen-complex; and c) substituting said at least oneamino acid residue identified in step b) with an amino acid having alarger side chain volume.
 2. The method of claim 1, wherein the antibodybinds to the same or overlapping epitope than an antibody characterizedby a VH of SEQ ID NO:01 and a VL of SEQ ID NO:02.
 3. The method of claim1, wherein the antibody binds to a conformational epitope on a dimer ofVEGF-A121, wherein VEGF-A121 comprises an amino acid sequence of SEQ IDNO: 36, wherein the epitope comprises: in one of the individualVEGF-A121 molecules within the VEGF dimer amino acids F17, M18, D19,Y21, Q22, R23, Y25, H27, P28, 129, E30, M55, N62, L66, N100, K101, C102,E103, C104, R105 and P106; and in the other one of the individualVEGF-A121 molecules within the VEGF dimer amino acids E30, K48, M81 andQ87.
 4. The method of claim 1, wherein the amino acid having a largerside chain volume is an aromatic amino acid.
 5. The method of claim 1,wherein the at least one substituted amino acid residue is located inthe heavy chain variable domain of said antibody.
 6. The method of claim1, wherein the at least one substituted amino acid residue is locatedwithin a heavy chain CDR of said antibody.
 7. The method of claim 1,wherein the at least one substituted amino acid residue is locatedwithin H-CDR2 of said antibody.
 8. The method of claim 1, wherein the atleast one substituted amino acid residue replaced is located within aheavy chain FR of said antibody.
 9. The method of claim 1, wherein theat least one substituted amino acid residue is located within H-FR3 ofsaid antibody.
 10. The method of claim 1, wherein the antibodycomprises: comprises (a) CDR-H1 comprising the amino acid sequence ofSEQ ID NO:03; (b) CDR-H2 comprising the amino acid sequence of SEQ IDNO:04; (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO:05;(d) CDR-L1 comprising the amino acid sequence of SEQ ID NO:06; (e)CDR-L2 comprising the amino acid sequence of SEQ ID NO:07; and (f)CDR-L3 comprising the amino acid sequence of SEQ ID NO:08.
 11. Themethod of claim 1, wherein the antibody comprises a VH comprising SEQ IDNO:01 and a VL comprising SEQ ID NO:02.
 12. An antibody that binds toVEGF produced by the method of claim
 1. 13. The antibody according toclaim 12, wherein binding of the antibody to VEGF significantly inhibitsVEGF-binding to VEGF receptor VEGF-R2 without significantly inhibitingVEGF-binding to VEGF receptor VEGF-R1.
 14. An isolated nucleic acidencoding the antibody of claim
 10. 15. A host cell comprising thenucleic acid of claim 14.